The present invention relates to methods of selectively derivatizing taxanes such as paclitaxel at the 2xe2x80x2-position thereof with amino acids and the like. The invention also relates to polymer conjugates made therewith.
Various plant alkaloids such as the vinka derivatives vinblastine and vincristine, camptothecin and paclitaxel have been shown to have potent anti-cancer effects. Such alkaloids are often poorly soluble. Indeed, because paclitaxel is so poorly soluble, the commercially available formulation for injection or I.V. infusion includes the solubilizer Cremophore EL. Cremophore, however, can be toxic. It is associated with idiosyncratic histamine release and anaphylactic reactions. Alternatives have therefore been sought.
One solution to improve solubility has been to provide amino acid derivatives of the desired anticancer alkaloids. For example, U.S. Pat. No. 4,943,579 discloses certain amino acid derivatives of camptothecin as having improved water solubility. The camptothecin is first converted to the chloroacetate using chloroacetic anhydride, pyridine and DMAP. The chloroacetate is then converted to the iodoacetate before being finally converted into the amino acid ester using a secondary amine. The stability of the final product, (a salt thereof) is only reported in terms of hydrolysis in plasma.
Amino acid derivatives of paclitaxel have also been disclosed. See, for example, U.S. Pat. No. 4,960,790 to Stella, et al., which discloses various 2xe2x80x2- and 7-protected amino acid paclitaxels. According to Stella, the reaction of the alkylated or protected amino acid is conducted in the presence of a condensing reagent, optionally with a catalyst, preferably at room temperature. Mentioned condensing reagents include carbodiimides, such as dicyclohexyl carbodiimide (DCC), while the catalysts mentioned include 4-dimethylamino-pyridine (DMAP) and pyridine. More importantly, the amino acid protecting groups employed include t-BOC, Fmoc or carbobenzyloxy (CBZ). Degradation of the final product and stereochemical modification are observed during deprotection. The problem is especially troublesome when synthesizing 2xe2x80x2-gly-paclitaxel. Deprotecting the 2xe2x80x2-gly-paclitaxel under acidic conditions makes purification and recovery of the free 2xe2x80x2-gly-paclitaxel almost impossible because of decomposition.
The use of formic acid to deprotect 2xe2x80x2-t-Boc amino acid taxanes has also been suggested. Shortcomings, however, have been associated with process as well. See, Mathew, A., et al. xe2x80x9cSynthesis and Evaluation of Some Water-Soluble Prodrugs and Derivatives of Taxol With Antitumor Activityxe2x80x9d, J. Med. Chem. 1992, 35, 145-151. First, the 2xe2x80x2-amino acid paclitaxel is produced in low yield, and a complicated purification must be employed for isolation. In addition, substantially complete rapid decomposition of the 2xe2x80x2-gly-paclitaxel derivative was still observed. Thus, further improvements are desirable.
Another process for providing 2xe2x80x2-amino acid paclitaxel derivatives includes using Fmoc protected amino acids. Acceptable yields of the 2xe2x80x2-amino acid paclitaxels are obtained after the protecting group is removed with an excess of piperidine. Although the free amino acid derivative is formed in the piperidine-containing mixture, substantial decomposition occurs during purification and isolation. The problem is particularly observed in the case of synthesizing 2xe2x80x2-gly-derivative.
A further refinement of the 2xe2x80x2-amino acid taxane synthesis proposed deblocking the Fmoc protecting group with DMAP rather than with piperidine at elevated temperature. While this has reduced the decomposition of the free 2xe2x80x2-amino acid taxane somewhat, further improvements have been sought.
Carpino et al. in J. Am. Chem. Soc. 1997, (119) pp 9915-9916, disclose the use of 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonbyl (hereinafter xe2x80x9cBsmocxe2x80x9d) as an alternative to Fmoc in peptide synthesis. Deprotection of Bsmoc amino acids allows the concurrent scavenging of the beta elimination products. There is no disclosure or suggestion of using the reagent in a process for attaching an amino acid or peptide to taxane derivatives, or that deprotecting of 2xe2x80x2-Bsmoc-amino acid taxanes with secondary amines could reduce or even overcome the problems associated with the use of other protected amino acids and deprotecting reagents.
In view of the foregoing, there is still a need for improving the processes employed for making stable amino acid esters of taxanes. The present invention addresses this need.
In one aspect of the invention, there is provided a method of preparing a 2xe2x80x2-substituted taxanes such as paclitaxel. The method includes: 
wherein:
R1 is selected from among phenyl, t-butoxy, isopropyloxy, propyloxy, xe2x80x94C(CH3)xe2x95x90CHxe2x80x94CH3, 2-naphthyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 2-methyl-1-propenyl, cyclopropyl, 3-furanyl, 3-thioethyl and 2-propenyl;
R2 is one of acetyl, xe2x80x94CH3, xe2x80x94CH2CH3 and xe2x80x94CHO;
R3 is selected from among acetyl, H and C1-6 alkyl;
R4 is selected from among H, F, C1-6 alkyl, xe2x80x94C(O)xe2x80x94CH2CH2CH2CH2CH3, xe2x80x94CH2SCH3, xe2x80x94SiEt3, xe2x80x94CH2OP(O)(OCH2Ph)2, CH3CH2C(O)xe2x80x94, xe2x80x94CH2O(CO)CH2N(CH2CH2)2NCH3, xe2x80x94CH2O(CO)CH2N(CH2CH3)2, xe2x80x94C(O)CH2N(CH3)2, xe2x80x94C(O)CH(CH3)NHCOOC(CH3)3; and
R5 is selected from among phenyl, 4-methoxyphenyl, 3,4-dimethoxyphenyl, 4-fluorophenyl, 4-trifluorotoluene, 2-furanyl, 2-thienyl, phenylethene, 2-furanyl-CHxe2x95x90CHxe2x80x94, (CH3)2CHCH2xe2x80x94, C6H11xe2x80x94CH2xe2x80x94, (CH3)2CHxe2x80x94, PhCH2CH2xe2x80x94, C6H11xe2x80x94CH2CH2xe2x80x94, CH3CH2CH2xe2x80x94, 4xe2x80x94Cl-phenyl-, 2-fluorophenyl-, 3-fluoro-phenyl- and 4-CH3-phenyl- with a compound of formula (II) 
wherein: 
L1 is a bifunctional group;
Y1 is selected from among O, S or NR7;
R6 and R7 are independently selected from among hydrogen, C1-6 alkyls, C3-19 branched alkyls, C3-8 cycloalkyls, C1-6 substituted alkyls, C3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C1-6 heteroalkyls, substituted C1-6 heteroalkyls, C1-6 alkoxy, phenoxy and C1-6 heteroalkoxy; and
J1 is OH or a leaving group;
under conditions sufficient to form a blocked intermediate of the formula (III) 
b) deprotecting the blocked intermediate with about an equimolar amount of a secondary amine, such as piperidine or 4-piperidinopiperidine, under conditions sufficient to form a compound of formula (IV): 
Another aspect of the invention includes reacting a compound of formula (IV), in situ, if desired, with an activated polymer of formula (Va):
R8xe2x80x94(L2)dxe2x80x94C(xe2x95x90Y2)xe2x80x94J2 or
(Vb) J2xe2x80x94C(xe2x95x90Y2)xe2x80x94(L2)dxe2x80x94R8xe2x80x94(L2)dxe2x80x94C(xe2x95x90Y2)xe2x80x94J2 to form a polymer conjugate of formula (VIa): 
or formula (VIb): 
wherein
R8 is a residue of a substantially non-antigenic polymer;
L2 is a bifunctional linker selected from among the same members of the group which comprise L1;
Y2 is selected from among O, S and R7a where R7a is selected from the same group which defines R7;
d is zero or one; and
J2 is OH or a leaving group.
In preferred aspects of this embodiment, the activated polymers are either mono- or bis PEG-CO2H.
The polymer conjugates can be used in the treatment of various taxane-sensitive conditions known to those of ordinary skill.
For purposes of the present invention, xe2x80x9cmild conditionsxe2x80x9d shall be understood to include, inter alia, temperatures around room temperature, short reaction times of about 1-2 hours, and non-molar excess of deprotective reagents.
For purposes of the present invention, the term xe2x80x9cresiduexe2x80x9d shall be understood to mean that portion of a compound, to which it refers, that remains after it has undergone a substitution reaction in which the polymeric prodrug carrier portion has been attached.
For purposes of the present invention, the term xe2x80x9cpolymeric residuexe2x80x9d or xe2x80x9cPEG residuexe2x80x9d shall each be understood to mean that portion of the polymer or PEG which remains after it has undergone a reaction with a biologically active compound.
For purposes of the present invention, the term xe2x80x9calkylxe2x80x9d shall be understood to include straight, branched, substituted, e.g. halo-, alkoxy-, nitro-, C1-12 alkyls, C3-8 cycloalkyls or substituted cycloalkyls, etc.
For purposes of the present invention, the term xe2x80x9csubstitutedxe2x80x9d shall be understood to include adding or replacing one or more atoms contained within a functional group or compound with one or more different atoms.
For purposes of the present invention, substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyls; substituted alkenyls include carboxyalkenyls, aminoalkenyls, dialkenylaminos, hydroxyalkenyls and mercaptoalkenyls; substituted alkynyls include carboxyalkynyls, aminoalkynyls, dialkynylaminos, hydroxyalkynyls and mercaptoalkynyls; substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as napthyl; substituted aryls include moieties such as 3-bromo-phenyl; aralkyls include moieties such as toluyl; heteroalkyls include moieties such as ethylthiophene; substituted heteroalkyls include moieties such as 3-methoxy-thiophene; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo- shall be understood to include fluoro, chloro, iodo and bromo.
The term xe2x80x9csufficient amountsxe2x80x9d for purposes of the present invention shall mean an amount which achieves a therapeutic effect as such effect is understood by those of ordinary skill in the art.
For purposes of the present invention, xe2x80x9ceffectively non-antigenicxe2x80x9d and xe2x80x9csubstantially non-antigenicxe2x80x9d shall be understood to include all polymeric materials understood in the art as being substantially non-toxic and not eliciting an appreciable immune response in mammals.
For purposes of the present invention, a xe2x80x9cpositive integerxe2x80x9d shall be understood to mean a positive whole number, preferably from about 1 to 6 and more preferably 1 or 2.
As a result of the present invention, there are provided improved processes for preparing amino acid esters of taxanes. The compounds made with the process of the present invention find utility, for example as pharmacological agents and as important intermediates in the formation of 2xe2x80x2-taxane polymer conjugates. The use of Bsmoc and related amino-protecting groups allow the artisan to form stable 2xe2x80x2-substituted taxane end products in high yield and in economical fashion. The use of Bsmoc protected amino acids allows the artisan to make 2xe2x80x2-protected amino acid taxanes in relatively high yields with minimal purification being required. Without wishing to be bound by theory, the desired products are obtained with minimal degradation because the Bsmoc deprotection of the amino acid taxane can be achieved under mild conditions with about an equimolar, rather than excess, amount of the secondary amine base. These conditions allow the desired free amino acid taxane to be isolated and recovered in quantitative yields and minimal decomposition.
A further advantage of using the Bsmoc-based processes of the present invention is the fact that the deprotection reaction acts simultaneously as a scavenging reaction to remove the Beta elimination product. Thus, purification and even in situ PEGylation are quite economical when compared to prior art techniques. Other and further advantages will be apparent to those of ordinary skill in view of the following description and appended claims.